Genome-Scale Metabolic Reconstruction, Non-Targeted LC-QTOF-MS Based Metabolomics Data, and Evaluation of Anticancer Activity of Cannabis sativa Leaf Extracts.

Over the past decades, Colombia has suffered complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations. Considerable effort has been made in the regulation of illicit crops, predominantly Cannabis sativa, leading to advances such as the legalization of medical cannabis and its derivatives, the improvement of crops, and leaving an open window to the development of scientific knowledge to explore alternative uses. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites. Some of the most relevant due to their anticancer properties, besides cannabinoids, are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, and phenolic compounds. However, despite the increase in scientific research on the subject, it is necessary to study the primary and secondary metabolism of the plant and to identify key pathways that explore its great metabolic potential. For this purpose, a genome-scale metabolic reconstruction of C. sativa is described and contextualized using LC-QTOF-MS metabolic data obtained from the leaf extract from plants grown in the region of Pesca-Boyaca, Colombia under greenhouse conditions at the Clever Leaves facility. A compartmentalized model with 2101 reactions and 1314 metabolites highlights pathways associated with fatty acid biosynthesis, steroids, and amino acids, along with the metabolism of purine, pyrimidine, glucose, starch, and sucrose. Key metabolites were identified through metabolomic data, such as neurine, cannabisativine, cannflavin A, palmitoleic acid, cannabinoids, geranylhydroquinone, and steroids. They were analyzed and integrated into the reconstruction, and their potential applications are discussed. Cytotoxicity assays revealed high anticancer activity against gastric adenocarcinoma (AGS), melanoma cells (A375), and lung carcinoma cells (A549), combined with negligible impact against healthy human skin cells.

Tailoring Magnetite-Nanoparticle-Based Nanocarriers for Gene Delivery: Exploiting CRISPRa Potential in Reducing Conditions.

Gene delivery has emerged as a promising alternative to conventional treatment approaches, allowing for the manipulation of gene expression through gene insertion, deletion, or alteration. However, the susceptibility of gene delivery components to degradation and challenges associated with cell penetration necessitate the use of delivery vehicles for effective functional gene delivery. Nanostructured vehicles, such as iron oxide nanoparticles (IONs) including magnetite nanoparticles (MNPs), have demonstrated significant potential for gene delivery applications due to their chemical versatility, biocompatibility, and strong magnetization. In this study, we developed an ION-based delivery vehicle capable of releasing linearized nucleic acids (tDNA) under reducing conditions in various cell cultures. As a proof of concept, we immobilized a CRISPR activation (CRISPRa) sequence to overexpress the pink1 gene on MNPs functionalized with polyethylene glycol (PEG), 3-[(2-aminoethyl)dithio]propionic acid (AEDP), and a translocating protein (OmpA). The nucleic sequence (tDNA) was modified to include a terminal thiol group and was conjugated to AEDP's terminal thiol via a disulfide exchange reaction. Leveraging the natural sensitivity of the disulfide bridge, the cargo was released under reducing conditions. Physicochemical characterizations, including thermogravimetric analysis (TGA) and Fourier-transform infrared (FTIR) spectroscopy, confirmed the correct synthesis and functionalization of the MNP-based delivery carriers. The developed nanocarriers exhibited remarkable biocompatibility, as demonstrated by the hemocompatibility, platelet aggregation, and cytocompatibility assays using primary human astrocytes, rodent astrocytes, and human fibroblast cells. Furthermore, the nanocarriers enabled efficient cargo penetration, uptake, and endosomal escape, with minimal nucleofection. A preliminary functionality test using RT-qPCR revealed that the vehicle facilitated the timely release of CRISPRa vectors, resulting in a remarkable 130-fold overexpression of pink1. We demonstrate the potential of the developed ION-based nanocarrier as a versatile and promising gene delivery vehicle with potential applications in gene therapy. The developed nanocarrier is capable of delivering any nucleic sequence (up to 8.2 kb) once it is thiolated using the methodology explained in this study. To our knowledge, this represents the first MNP-based nanocarrier capable of delivering nucleic sequences under specific reducing conditions while preserving functionality.

Multifunctional magnetoliposomes as drug delivery vehicles for the potential treatment of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD.

Unlocking cellular barriers: silica nanoparticles and fullerenol conjugated cell-penetrating agents for enhanced intracellular drug delivery.

The limited delivery of cargoes at the cellular level is a significant challenge for therapeutic strategies due to the presence of numerous biological barriers. By immobilizing the Buforin II (BUF-II) peptide and the OmpA protein on magnetite nanoparticles, a new family of cell-penetrating nanobioconjugates was developed in a previous study. We propose in this study to extend this strategy to silica nanoparticles (SNPs) and silanized fullerenol (F) as nanostructured supports for conjugating these potent cell-penetrating agents. The same molecule conjugated to distinct nanomaterials may interact with subcellular compartments differently. On the obtained nanobioconjugates (OmpA-SNPs, BUF-II-PEG12-SNPs, OmpA-F, and BUF-II-PEG12-F), physicochemical characterization was performed to evaluate their properties and confirm the conjugation of these translocating agents on the nanomaterials. The biocompatibility, toxicity, and internalization capacity of nanobioconjugates in Vero cells and THP-1 cells were evaluated in vitro. Nanobioconjugates had a high internalization capacity in these cells without affecting their viability, according to the findings. In addition, the nanobioconjugates exhibited negligible hemolytic activity and a low tendency to induce platelet aggregation. In addition, the nanobioconjugates exhibited distinct intracellular trafficking and endosomal escape behavior in these cell lines, indicating their potential for addressing the challenges of cytoplasmic drug delivery and the development of therapeutics for the treatment of lysosomal storage diseases. This study presents an innovative strategy for conjugating cell-penetrating agents using silica nanoparticles and silanized fullerenol as nanostructured supports, which has the potential to enhance the efficacy of cellular drug delivery.

Formulation of a novel antibacterial topical treatment based on Magnetite-Buforin-II-silver nanobioconjugates.

Community acquired infections caused by Meticillin-resistant Staphylococcus aureus (MRSA) have become a growing concern due to its impact on the world public health. This microorganism is a commonly spreading pathogen associated predominantly with skin infections and connected to other more severe conditions (septic shock, and generalized infection). The lack of highly effective antibiotics and treatments to control skin infections with S. aureus has led to the search of novel therapies using alternative agents such as antimicrobial peptides (AMPs). In order to obtain a viable administration route to counteract superficial skin infections (impetigo, abscesses, furuncles, and cellulitis), a topical formulation based on Magnetite-Buforin-II-silver nanobioconjugates as active antibacterial agents was designed by their dispersion in O/W concentrated emulsions. The prepared topical characterization indicated that O/W emulsions were stable in time, the droplets size remained within the appropriate values (∼1 µm) and their rheological properties, such as pseudoplastic and shear-thinning behavior, remained unchanged for up to 3 months. Additionally, hemolysis and platelet aggregation tests were acceptable (i.e., 14.72 ± 2.62% and 8.06 ± 2.90%, respectively) in compliance with the ISO-10993 standard. Furthermore, the treatment reduced significantly (p < 0.0001) the growth of both clinical isolated MRSA and wild Type S. aureus strains as evidenced by the contact diffusion method. These results are important in the context of proposing new alternatives that allow manage effectively the threat posed by the antibiotic resistant bacterial strains, which jeopardize the lives of thousands of people every year.

Assessing cellular internalization and endosomal escape abilities of novel BUFII-Graphene oxide nanobioconjugates.

Cell-penetrating agents based on functionalized nanoplatforms have emerged as a promising approach for developing more efficient and multifunctional delivery vehicles for treating various complex diseases that require reaching different intracellular compartments. Our previous work has shown that achieving full cellular coverage and high endosomal escape rates is possible by interfacing magnetite nanoparticles with potent translocating peptides such as Buforin II (BUF-II). In this work, we extended such an approach to two graphene oxide (GO)-based nanoplatforms functionalized with different surface chemistries to which the peptide molecules were successfully conjugated. The developed nanobioconjugates were characterized via spectroscopic (FTIR, Raman), thermogravimetric, and microscopic (SEM, TEM, and AFM) techniques. Moreover, biocompatibility was assessed via standardized hemocompatibility and cytotoxicity assays in two cell lines. Finally, cell internalization and coverage and endosomal escape abilities were estimated with the aid of confocal microscopy analysis of colocalization of the nanobioconjugates with Lysotracker Green®. Our findings showed coverage values that approached 100% for both cell lines, high biocompatibility, and endosomal escape levels ranging from 30 to 45% and 12-24% for Vero and THP-1 cell lines. This work provides the first routes toward developing the next-generation, carbon-based, cell-penetrating nanovehicles to deliver therapeutic agents. Further studies will be focused on elucidating the intracellular trafficking pathways of the nanobioconjugates to reach different cellular compartments.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

Reduced Graphene Oxide-Extracellular Matrix Scaffolds as a Multifunctional and Highly Biocompatible Nanocomposite for Wound Healing: Insights into Characterization and Electroconductive Potential.

The development of novel regenerative technologies based on the implementation of natural extracellular matrix (ECM), or individual components of ECM combined with multifunctional nanomaterials such as graphene oxide and reduced graphene oxide, has demonstrated remarkable results in wound healing and tissue engineering. However, the synthesis of these nanocomposites involves great challenges related to maintaining the biocompatibility with a simultaneous improvement in their functionalities. Based on that, in this research we developed novel nanoengineered ECM-scaffolds formed by mixing small intestinal submucosa (SIS) with graphene oxide (GO)/reduced graphene oxide (rGO) to improve electrical conductivity while maintaining remarkable biocompatibility. For this, decellularized SIS was combined with GO to form the scaffold precursor for subsequent lyophilization, chemically crosslinking and in situ reduction. The obtained GO and rGO were characterized via Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), electrical conductivity testing and atomic force microscopy (AFM). The results confirm the suitable synthesis of GO, the effective reduction to rGO and the significant increase in the electrical conductivity (more than four orders of magnitude higher than bare GO). In addition, the graphene oxide/reduced graphene oxide-SIS scaffolds were characterized via Raman spectroscopy, FTIR, TGA, SEM, porosity assay (higher than 97.5% in all cases) and protein secondary structural analysis. Moreover, the biocompatibility of scaffolds was studied by standardized assays of hemolysis activity (less than 0.5%), platelet activation and deposition, and cell viability in Vero, HaCat and HFF-1 cells (higher than 90% for all evaluated cell lines on the different scaffolds). The obtained results confirm the remarkable biocompatibility, as supported by high hemocompatibility, low cytotoxicity and no negative impact on platelet activation and deposition. Finally, structural characteristics such as pore size and interconnectivity as well as superior cell attachment abilities also corroborated the potential of the developed nanoengineered ECM-scaffolds as a multifunctional nanoplatform for application in regenerative medicine and tissue engineering.

Novel Photo- and Thermo-Responsive Nanocomposite Hydrogels Based on Functionalized rGO and Modified SIS/Chitosan Polymers for Localized Treatment of Malignant Cutaneous Melanoma.

Melanoma is an aggressive type of skin cancer that accounts for over 75% of skin cancer deaths despite comprising less than 5% of all skin cancers. Despite promising improvements in surgical approaches for melanoma resection, the survival of undetectable microtumor residues has remained a concern. As a result, hyperthermia- and drug-based therapies have grown as attractive techniques to target and treat cancer. In this work, we aim to develop a stimuli-responsive hydrogel based on chitosan methacrylate (ChiMA), porcine small intestine submucosa methacrylate (SISMA), and doxorubicin-functionalized reduced graphene oxide (rGO-DOX) that eliminates microtumor residues from surgically resected melanoma through the coupled effect of NIR light-induced photothermal therapy and heat-induced doxorubicin release. Furthermore, we developed an in silico model to optimize heat and mass transport and evaluate the proposed chemo/photothermal therapy in vitro over melanoma cell cultures.

Novel antibacterial hydrogels based on gelatin/polyvinyl-alcohol and graphene oxide/silver nanoconjugates: formulation, characterization, and preliminary biocompatibility evaluation.

Antibiotic resistance has become a major public health problem generated by their excessive and inappropriate use. This is worrisome because multiple microbial infections that could traditionally be treated without major complications are now considerably challenging to treat. In this regard, research in this field has been focused on searching for new molecules capable of arresting these microbial infections with high effectiveness, including antimicrobial peptides (AMP) and various nanomaterials. Here, we proposed a novel topical hydrogel treatment based on a polymeric network of gelatin-polyvinyl alcohol-hyaluronic acid encapsulating a graphene oxide (GO) nanoconjugate on which silver nanoparticles (Ag NPs) have been grown. This treatment is intended to be stable, biocompatible, non-toxic, pleasant to skin contact, provide bioavailability of the active agent for a prolonged period in the affected skin area where its application is required and inhibit microbial growth effectively. The nanocomposite hydrogels were characterized in terms of microstructure, thermal resistance, rheological behavior, particle size distribution, texture profile and physical stability, as well as a one-month accelerated stability study. The satisfactory results in terms of physical chemistry, stability on storage modulus (G'), TSI values, and microstructure allowed choosing some points of the experimental design to encapsulate the GO-Ag NPs nanoconjugates. The biological evaluation of these nanocomposites showed that the treatments are biocompatible as they have a very low hemolytic effect (less than 5%) and a moderate platelet aggregating capacity (35%-45%). Finally, 100% of bacterial growth was inhibited by the action of the topical nanocomposite hydrogel treatments. These results led to affirm that these treatments can have an excellent performance in this application as well as in wound healing and dressing, bioadhesives, tissue engineering, and other biomedical applications.

Rational Discovery of Antimicrobial Peptides by Means of Artificial Intelligence.

Antibiotic resistance is a worldwide public health problem due to the costs and mortality rates it generates. However, the large pharmaceutical industries have stopped searching for new antibiotics because of their low profitability, given the rapid replacement rates imposed by the increasingly observed resistance acquired by microorganisms. Alternatively, antimicrobial peptides (AMPs) have emerged as potent molecules with a much lower rate of resistance generation. The discovery of these peptides is carried out through extensive in vitro screenings of either rational or non-rational libraries. These processes are tedious and expensive and generate only a few AMP candidates, most of which fail to show the required activity and physicochemical properties for practical applications. This work proposes implementing an artificial intelligence algorithm to reduce the required experimentation and increase the efficiency of high-activity AMP discovery. Our deep learning (DL) model, called AMPs-Net, outperforms the state-of-the-art method by 8.8% in average precision. Furthermore, it is highly accurate to predict the antibacterial and antiviral capacity of a large number of AMPs. Our search led to identifying two unreported antimicrobial motifs and two novel antimicrobial peptides related to them. Moreover, by coupling DL with molecular dynamics (MD) simulations, we were able to find a multifunctional peptide with promising therapeutic effects. Our work validates our previously proposed pipeline for a more efficient rational discovery of novel AMPs.

Translocating Peptides of Biomedical Interest Obtained from the Spike (S) Glycoprotein of the SARS-CoV-2.

At the beginning of 2020, the pandemic caused by the SARS-CoV-2 virus led to the fast sequencing of its genome to facilitate molecular engineering strategies to control the pathogen's spread. The spike (S) glycoprotein has been identified as the leading therapeutic agent due to its role in localizing the ACE2 receptor in the host's pulmonary cell membrane, binding, and eventually infecting the cells. Due to the difficulty of delivering bioactive molecules to the intracellular space, we hypothesized that the S protein could serve as a source of membrane translocating peptides. AHB-1, AHB-2, and AHB-3 peptides were identified and analyzed on a membrane model of DPPC (dipalmitoylphosphatidylcholine) using molecular dynamics (MD) simulations. An umbrella sampling approach was used to quantify the energy barrier necessary to cross the boundary (13.2 to 34.9 kcal/mol), and a flat-bottom pulling helped to gain a deeper understanding of the membrane's permeation dynamics. Our studies revealed that the novel peptide AHB-1 exhibited comparable penetration potential of already known potent cell-penetrating peptides (CPPs) such as TP2, Buforin II, and Frenatin 2.3s. Results were confirmed by in vitro analysis of the peptides conjugated to chitosan nanoparticles, demonstrating its ability to reach the cytosol and escape endosomes, while maintaining high biocompatibility levels according to standardized assays.

Microfluidic Synthesis and Purification of Magnetoliposomes for Potential Applications in the Gastrointestinal Delivery of Difficult-to-Transport Drugs.

Magnetite nanoparticles (MNPs) have gained significant attention in several applications for drug delivery. However, there are some issues related to cell penetration, especially in the transport of cargoes that show limited membrane passing. A widely studied strategy to overcome this problem is the encapsulation of the MNPs into liposomes to form magnetoliposomes (MLPs), which are capable of fusing with membranes to achieve high delivery rates. This study presents a low-cost microfluidic approach for the synthesis and purification of MLPs and their biocompatibility and functional testing via hemolysis, platelet aggregation, cytocompatibility, internalization, and endosomal escape assays to determine their potential application in gastrointestinal delivery. The results show MLPs with average hydrodynamic diameters ranging from 137 ± 17 nm to 787 ± 45 nm with acceptable polydispersity index (PDI) values (below 0.5). In addition, we achieved encapsulation efficiencies between 20% and 90% by varying the total flow rates (TFRs), flow rate ratios (FRRs), and MNPs concentration. Moreover, remarkable biocompatibility was attained with the obtained MLPs in terms of hemocompatibility (hemolysis below 1%), platelet aggregation (less than 10% with respect to PBS 1×), and cytocompatibility (cell viability higher than 80% in AGS and Vero cells at concentrations below 0.1 mg/mL). Additionally, promising delivery results were obtained, as evidenced by high internalization, low endosomal entrapment (AGS cells: PCC of 0.28 and covered area of 60% at 0.5 h and PCC of 0.34 and covered area of 99% at 4 h), and negligible nuclear damage and DNA condensation. These results confirm that the developed microfluidic devices allow high-throughput production of MLPs for potential encapsulation and efficient delivery of nanostructured cell-penetrating agents. Nevertheless, further in vitro analysis must be carried out to evaluate the prevalent intracellular trafficking routes as well as to gain a detailed understanding of the existing interactions between nanovehicles and cells.

Recent Advances on Stimuli-Responsive Hydrogels Based on Tissue-Derived ECMs and Their Components: Towards Improving Functionality for Tissue Engineering and Controlled Drug Delivery.

Due to their highly hydrophilic nature and compositional versatility, hydrogels have assumed a protagonic role in the development of physiologically relevant tissues for several biomedical applications, such as in vivo tissue replacement or regeneration and in vitro disease modeling. By forming interconnected polymeric networks, hydrogels can be loaded with therapeutic agents, small molecules, or cells to deliver them locally to specific tissues or act as scaffolds for hosting cellular development. Hydrogels derived from decellularized extracellular matrices (dECMs), in particular, have gained significant attention in the fields of tissue engineering and regenerative medicine due to their inherently high biomimetic capabilities and endowment of a wide variety of bioactive cues capable of directing cellular behavior. However, these hydrogels often exhibit poor mechanical stability, and their biological properties alone are not enough to direct the development of tissue constructs with functional phenotypes. This review highlights the different ways in which external stimuli (e.g., light, thermal, mechanical, electric, magnetic, and acoustic) have been employed to improve the performance of dECM-based hydrogels for tissue engineering and regenerative medicine applications. Specifically, we outline how these stimuli have been implemented to improve their mechanical stability, tune their microarchitectural characteristics, facilitate tissue morphogenesis and enable precise control of drug release profiles. The strategic coupling of the bioactive features of dECM-based hydrogels with these stimulation schemes grants considerable advances in the development of functional hydrogels for a wide variety of applications within these fields.

Antioxidant and Neuroprotective Properties of Non-Centrifugal Cane Sugar and Other Sugarcane Derivatives in an In Vitro Induced Parkinson's Model.

Non-centrifugal cane sugar (NCS) is a traditional sweetener in most sugarcane regions of the world. In Colombia, this product has a socio-economic importance due to the extensive cultivation area and the high consumption rate per capita. NCS traditional processing involves consecutive stages of thermal processing that begin with juice extraction, clarification, evaporation, and finish with syrup crystallization into a solid commercial product, identified as NCS. Sugarcane is known to have a natural content of polyphenols, amino acids, vitamins, minerals, and complex sugars, some of which are reported as antioxidant and antiproliferative agents thought to be responsible for the product's bioactive profile. There is evidence to suggest that traditional thermal processing to obtain NCS leads to a considerable decrease in the contents of these bioactive compounds, mainly due to uncontrolled process variables such as temperature. Accordingly, the aim of this study was to assess and compare the bioactivity of sugarcane (SC) derivatives produced under controlled thermal conditions versus the traditional method. To achieve this goal, we evaluated the cytotoxic, antioxidant, and neuroprotective effects of varying concentrations of SC derivatives in an in vitro induced Parkinson's model. Results demonstrate non-cytotoxic activity on the cellular model by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LDH assays, even at the highest tested concentration of 8 mg/mL, for all SC derivatives. The effect of SC derivatives on the induced oxidative stress model showed a biological reversion and recovering effect of the mitochondrial membrane potential and a halting of the progress into the early apoptosis phase. In conclusion, we demonstrated that the bioactive compounds present in SC derivatives obtained by a process under controlled temperature conditions are largely preserved, and even their biological activities are enhanced compared with SC derivatives obtained by the traditional thermal evaporation of SC-juice.

Gelatin-Graphene Oxide Nanocomposite Hydrogels for Kluyveromyces lactis Encapsulation: Potential Applications in Probiotics and Bioreactor Packings.

Nutraceutical formulations based on probiotic microorganisms have gained significant attention over the past decade due to their beneficial properties on human health. Yeasts offer some advantages over other probiotic organisms, such as immunomodulatory properties, anticancer effects and effective suppression of pathogens. However, one of the main challenges for their oral administration is ensuring that cell viability remains high enough for a sustained therapeutic effect while avoiding possible substrate inhibition issues as they transit through the gastrointestinal (GI) tract. Here, we propose addressing these issues using a probiotic yeast encapsulation strategy, Kluyveromyces lactis, based on gelatin hydrogels doubly cross-linked with graphene oxide (GO) and glutaraldehyde to form highly resistant nanocomposite encapsulates. GO was selected here as a reinforcement agent due to its unique properties, including superior solubility and dispersibility in water and other solvents, high biocompatibility, antimicrobial activity, and response to electrical fields in its reduced form. Finally, GO has been reported to enhance the mechanical properties of several materials, including natural and synthetic polymers and ceramics. The synthesized GO-gelatin nanocomposite hydrogels were characterized in morphological, swelling, mechanical, thermal, and rheological properties and their ability to maintain probiotic cell viability. The obtained nanocomposites exhibited larger pore sizes for successful cell entrapment and proliferation, tunable degradation rates, pH-dependent swelling ratio, and higher mechanical stability and integrity in simulated GI media and during bioreactor operation. These results encourage us to consider the application of the obtained nanocomposites to not only formulate high-performance nutraceuticals but to extend it to tissue engineering, bioadhesives, smart coatings, controlled release systems, and bioproduction of highly added value metabolites.

Graphene Oxide-Embedded Extracellular Matrix-Derived Hydrogel as a Multiresponsive Platform for 3D Bioprinting Applications.

Decellularized extracellular matrices (dECMs) have shown enormous potential for the biofabrication of tissues due to their biomimetic properties that promote enhanced cellular interaction and tissue regeneration. However, biofabrication schemes requiring electrostimulation pose an additional constraint due to the insulating properties of natural materials. Here, we propose a methacryloyl-modified decellularized small intestine submucosa (SISMA) hydrogel, embedded with graphene oxide (GO) nanosheets, for extrusion-based 3D bioprinting applications that require electrostimulation. Methacryloyl biochemical modification is performed to enhance the mechanical stability of dECM constructs by mediating photo-crosslinking reactions, and a multistep fabrication scheme is proposed to harness the bioactive and hydrophilic properties of GO and electroconductive properties of reduced GO. For this, GO was initially dispersed in SISMA hydrogels by exploiting its hydrophilicity and protein adsorption capabilities, and in situ reduction was subsequently performed to confer electroconductive abilities. SISMA-GO composite hydrogels were successfully prepared with enhanced structural characteristics, as shown by the higher crosslinking degree and increased elastic response upon blue-light exposure. Moreover, GO was homogeneously dispersed without affecting photocrosslinking reactions and hydrogel shear-thinning properties. Human adipose-derived mesenchymal stem cells were successfully bioprinted in SISMA-GO with high cell viability after 1 week and in situ reduction of GO during this period enhanced the electrical conductivity of these nanostructures. This work demonstrates the potential of SISMA-GO bioinks as bioactive and electroconductive scaffolds for electrostimulation applications in tissue engineering and regenerative medicine.

Preparation and Characterization of an Injectable and Photo-Responsive Chitosan Methacrylate/Graphene Oxide Hydrogel: Potential Applications in Bone Tissue Adhesion and Repair.

As life expectancy continues to increase, the inevitable weakening and rupture of bone tissue have grown as concerns in the medical community, thus leading to the need for adhesive materials suitable for bone repair applications. However, current commercially available adhesives face certain drawbacks that prevent proper tissue repair, such as low biocompatibility, poor adhesion to wet surfaces, and the need for high polymerization temperatures. This work aims to develop an injectable and photo-responsive chitosan methacrylate/graphene oxide (ChiMA/GO) adhesive nanocomposite hydrogel of high biocompatibility that is easy to apply by simple extrusion and that offers the possibility for in situ polymer and physiological temperatures. The nanocomposite was thoroughly characterized spectroscopically, microscopically, rheologically, thermally, and through mechanical, textural, and biological assays to fully evaluate its correct synthesis and functionalization and its performance under physiological conditions that mimic those observed in vivo. In addition, a finite element analysis (FEA) simulation was used to evaluate its performance in femur fractures. Results suggest the material's potential as a bioadhesive, as it can polymerize at room temperature, shows superior stability in physiological media, and is capable of withstanding loads from body weight and movement. Moreover, the material showed remarkable biocompatibility as evidenced by low hemolytic and intermediate platelet aggregation tendencies, and high cytocompatibility when in contact with osteoblasts. The comprehensive studies presented here strongly suggest that the developed hydrogels are promising alternatives to conventional bone adhesives that might be further tested in vivo in the near future.

Hospitalisation in high-risk pregnancy patients: is prenatal attachment affected?

Objective: To compare prenatal attachment in women hospitalised due to high-risk pregnancy with prenatal attachment in non-hospitalised patients. To describe the impact of social support, socio-demographic factors and the nature of the pregnancy on prenatal attachment, anxiety and depression. Study Design: An exploratory, cross-sectional and descriptive study utilising the Maternal Antenatal Attachment Scale, the Edinburgh Postnatal Depression Scale and the State-Trait Anxiety Inventory. The sample comprised 80 hospitalised and 88 non-hospitalised patients. Result: No difference in prenatal attachment was found between the two groups. The hospitalised group presented higher levels of depressive symptomatology and anxiety. Social support had a significant effect on the hospitalised group, improving attachment quality. Conclusion: Incorporation of members of the patient's support network may help to improve quality of prenatal attachment during hospitalisation. Detection and treatment of anxiety and/or depression in hospitalised patients is recommended given their impact on the mental health of mother and baby.